Combinatorial Chemistry & High Throughput Screening - Volume 24, Issue 3, 2021

Background: Considering the low ocular bioavailability of conventional formulations used for ocular bacterial infection treatment, there is a need to design efficient novel drug delivery systems that may enhance precorneal retention time and corneal permeability. Aim and Objective: The current research focuses on developing nanosized and non-toxic Eudragit® RL 100 and Kollidon® SR nanoparticles loaded with moxifloxacin hydrochloride (MOX) for its prolonged release to be promising for effective ocular delivery. Methods: In this study, MOX incorporation was carried out by spray drying method aiming ocular delivery. In vitro characteristics were evaluated in detail with different methods. Results: MOX was successfully incorporated into Eudragit® RL 100 and Kollidon® SR polymeric nanoparticles by a spray-drying process. Particle size, zeta potential, entrapment efficiency, particle morphology, thermal, FTIR, NMR analyses and MOX quantification using HPLC method were carried out to evaluate the nanoparticles prepared. MOX loaded nanoparticles demonstrated nanosized and spherical shape while in vitro release studies demonstrated modified-release pattern, which followed the Korsmeyer-Peppas kinetic model. Following the successful incorporation of MOX into the nanoparticles, the formulation (MOX: Eudragit® RL 100, 1:5) (ERL-MOX 2) was selected for further studies because of its better characteristics like cationic zeta potential, smaller particle size, narrow size distribution and more uniform prolonged release pattern. Moreover, ERLMOX 2 formulation remained stable for 3 months and demonstrated higher cell viability values for MOX. Conclusion: In vitro characterization analyses showed that non-toxic, nano-sized and cationic ERL-MOX 2 formulation has the potential of enhancing ocular bioavailability.

Background: Terbinafine is an allylamine antifungal that is effective against many fungi, dermatophytes and moulds. Analytical methods are required for the determination of terbinafine in biological fluids to perform therapeutic drug monitoring and pharmacokinetic studies. Objective: The aim of this study was to develop and validate a novel and fast method combining dilute and shoot approach and high-performance liquid chromatography coupled with photodiode array detection for the determination of terbinafine in human urine. Methods: Chromatographic parameters including mobile phase composition, pH, flow rate and injection volume were assessed and optimized. The separation of terbinafine and naproxen (internal standard) was achieved within 3 min using a C18 core-shell column (Raptor ARC-18, 100 x 4.6 mm, 2.7 μm) under isocratic conditions. Samples were eluted from the column at the flow rate of 1.4 mL/min using a mobile phase containing 0.2% triethylamine in water (pH 3.4 with formic acid): acetonitrile (45:55, v/v). Results: The presented technique was linear in the range of 25-2000 ng/mL. Intra- and inter-day reproducibility at four quality control levels (25, 200, 750 and 1500 ng/mL) were less than 7%, with relative errors ranging from -5.40% to 5.91%. The limit of detection was 12.60 ng/mL. The developed method has three main advantages compared to existing methods: simplicity and greenness of sample preparation, use of core-shell column and short analysis time. Conclusion: The results of this study indicate that the combination of dilute and shoot approach and core-shell column can be regarded as an advantageous application for the fast determination of terbinafine in the urine.

Introduction: The alkyl esters of p-hydroxybenzoic acid at the C-4 position, “the parabens,” including methyl, ethyl, propyl, and butyl, are widely used as antimicrobial preservatives in foods, cosmetics, and pharmaceuticals. Official regulations on the use of these compounds make their analysis essential for the estimation of their exposure. Methods: On this basis, the presented study was realized to develop a simple, selective and cheap high-performance liquid chromatographic method for the quantitative determination of methylparaben, ethylparaben (EP), n-propyl paraben (NPP), isopropyl paraben (IPP), n-butyl paraben (NBP), isobutyl paraben (IBP) and benzyl paraben (BP) in pharmaceuticals and cosmetic products. Results: The chromatographic separation of the analytes was achieved under flow rate gradient elution conditions using a C18-bonded core-shell silica particle column (2.6 μm particle size, 150 × 3.0 mm from Phenomenex Co.). The samples were injected into the system as aliquots of 1.0 μL, and the compounds were detected by using a photodiode array detector set at 254 nm wavelength. With this technique, seven paraben derivatives can be determined in the concentration range of 250-2000 ng/mL. The recovery of the method is in the range of 99.95-13.84%, and the RSD is at a maximum value of 3.95%. Conclusion: The proposed method was fully validated and successfully applied to different pharmaceutical and cosmetic samples (n=16), including syrups, suspensions, oral sprays, gels, etc. At least one paraben derivative was detected in six samples and was determined quantitatively. The maximum amount of a paraben derivative found in the analyzed samples was 321.7 ng/mL, which was MP. To the best of our knowledge, this is the first LC method, which is applicable both on pharmaceutical and cosmetic samples.

Objective: The electrochemical analysis of ephedrine which is a sympathometric drug has been studied using poly(Nile blue A) modified glassy carbon electrodes, cyclic voltammetry, differential pulse voltammetry and square wave voltammetry. Methods: The modified electrodes were prepared by potential cycling electropolymerization of Nile blue A in 0.1 M phosphate buffer solution at pH 6.0. The redox behavior of ephedrine was investigated in different buffer solutions at pH values between 5.5 and 9.0. Results: Scan rate studies showed that the electron transfer reaction of ephedrine was diffusion controlled. A linear response was obtained between the peak current and the ephedrine concentration in the range of 0.6 to 100 μM with a limit of detection of 2.910^-3 μM for differential pulse voltammetry in Britton-Robinson buffer solution at pH 9.0. The linearity range of ephedrine in human urine was between 1.0 and 100 μM with a detection limit of 8.16 nM. Conclusion: The recovery studies in both pharmaceutical dosage forms and urine showed that the proposed method ensured good selectivity, precision and accuracy without any interference from inactive excipients.

Background: Electroanalytical methods are very functional to detect drugs in pharmaceuticals (tablets, syrups, suppositories, creams, and ointments) and biological samples. Objective: This study is aimed to make selective, sensitive, simple, fast, and low cost electrochemical analysis of expectorant drug guaifenesin in pharmaceuticals and serum samples. Methods: Differential pulse adsorptive stripping voltammetric method for determination of guaifenesin on a poly(acridine orange) modified glassy carbon electrode has been developed. Glassy carbon electrode was modified with electropolymerization of the acridine orange monomer for the sensitive determination of guaifenesin. Guaifenesin provided highly reproducible and welldefined irreversible oxidation peaks at +1.125 V and +1.128 V (vs. Ag/AgCl) in the selected supporting electrolyte and human serum samples, respectively. Results: Under optimized conditions, linear response of peak current on the concentration of guaifenesin has been obtained in the ranges of 2.00x10^-7 to 1.000^-4 M in Britton Robinson buffer solution at pH 7.0 and 4.000^-7 to 1.000^-4 M in serum samples. The precision of the method was detected by intraday and inter-day repeatability studies in the supporting electrolyte and serum samples media. Conclusion: The analytical applicability of the proposed method exhibited satisfying determination results for guaifenesin from pharmaceutical dosage forms (syrup) and human serum samples without any pre-separation procedures.

Introduction: In the present study, a sensitive and selective liquid chromatographytandem mass spectrometry (LC-MS/MS) method was described for the determination of ceftiofur (CEF) in cow milk and pharmaceutical preparations. CEF is an antibiotic compound, which is commonly used in the treatment of animal diseases such as respiratory system, soft tissue, and foot infections, as well as postpartum acute puerperal metritis. One of the critical features of CEF is its prescription while breastfeeding cows; in accordance, its quantitative estimation is essential to assess its residual amounts. Methods: In the method reported herein, after simple protein precipitation using acetonitrile, the pre-treated samples were introduced into an LC-MS/MS instrument equipped with a Chromolith® High-Resolution RP-18 series HPLC column (100 mm 4.6 mm from Merck KGaA, Germany). Electrospray ionization was employed as the ionization source in the triple-quadrupole tandem mass spectrometer. Results: For the calibration method using solvent-based standards, LOQ was 3.038 ng/mL, 12.15 ng/mL, and LOD was 1.215 ng/mL and 6.076 ng/mL for ESI+ and ESI- modes, respectively. On the other hand, for the method of matrix-matched standards, LOQ was 1.701 ng/mL, 10.13 ng/mL, and LOD was 0.486 ng/mL and 5.929 ng/mL for ESI+ and ESI- modes, respectively as obtained from signal to noise ratio. Conclusion: Applicability of both positive and negative ion modes was tested, and the analyte was detected via multiple reaction monitoring. The distorting effects of the milk matrix on the MS ionization and quantitation of CEF were overcome by using matrix-matched calibration for the first time.

Background: Bronchial asthma and chronic obstructive pulmonary disease (COPD) are among the most common chronic diseases. Roflumilast is a novel, potent, selective, and long-acting phosphodiesterase 4 (PDE-4) inhibitor for the treatment of bronchial asthma and COPD. It has anti-inflammatory effects, and it has been shown to reduce exacerbations and improve pulmonary function in patients with COPD. Although there have been some other analytical methodologies reported for the determination of roflumilast in pharmaceutical dosage forms, there has not yet been any electrochemical methodology proposed for determination of this unique active pharmaceutical ingredient in its dosage forms. Objective: The aim of this study was to develop an easily applied, selective, sensitive, accurate, and precise square-wave stripping voltammetric (SWSV) method for the determination of roflumilast in its pharmaceutical dosage forms. In addition, the electrochemical behavior of roflumilast was investigated. Methods: The proposed method was based on electrochemical reduction of roflumilast at a hanging mercury drop electrode (HMDE) in 0.1 M K2HPO4 and 0.1 M Na2B4O7 (1:1, v/v) buffer at pH 5.0. Two reduction peaks were observed at -1150 mV and -1260 mV with 30 s of accumulation time and -850 mV of accumulation potential time versus Ag/AgCl reference electrode. Results: The highest peak current values with the best peak definition were observed at a frequency of 50 Hz, scan increment of 5 mV, and pulse amplitude 25 mV. The proposed method was validated by evaluating validation parameters such as linearity, sensitivity, repeatability, accuracy, precision, selectivity, recovery, robustness, and ruggedness. A good linear correlation (r=0.9948) was obtained between the electrochemical response of roflumilast and its concentration in the range of 0.74-3.05 μg mL-1 under the optimum conditions. The obtained accuracy results were between 2.04% and -2.04% while the relative standard deviation of the results was at least 2.78% for intraday and inter-day studies. The mean recovery for the real applications was 100.63% ± 0.52. The electrochemical behavior of roflumilast was investigated by cyclic voltammetry. The cyclic voltammogram of roflumilast exhibited two peaks and the reduction reaction was reversible. Conclusion: This developed and validated SWSV method was applied successfully for the determination of roflumilast in tablet dosage form (Daxas®) to assess active roflumilast content. Since high- -performance liquid chromatography is a dominant technique in industry for quality control of active pharmaceutical ingredients, the finding in the present study demonstrated that square-wave stripping voltammetry could be easily utilized in routine applications to determine roflumilast content in its dosage forms.

Background: Hydrocephalus, a common brain disorder in children, can cause permanent brain damage. A timely diagnosis of this disorder is crucial. Objective: The aim of this study was to evaluate the levels of S-100, CK-18, and NSE brainspecific proteins in patients with hydrocephalus. We examined the levels of these proteins in the blood samples of hydrocephalic patients. Methods: The study was conducted on the hydrocephalus (n = 31) patients and a healthy control group (n = 30). A Receiver Operating Characteristic (ROC) curve was used to assess the validity of the NSE, CK-18, and S100B to differentiate between the hydrocephalus and the control groups. The suitability of the data to the normal distribution was tested with the Shapiro Wilk test, and the Student t-test was used to compare the characteristics of the normal distribution in two independent groups. The individuals in the hydrocephalus and control groups had similar values in terms of age, height, and weight. Results: It was observed that NSE, CK-18, and S100B mean values of the individuals in the hydrocephalus group were significantly higher than NSE, CK-18, and S100B mean values of the control group. Conclusion: Experiments have shown that the levels of these proteins increase significantly in hydrocephalus patients compared to the healthy group. These three parameters can be considered as important markers in the diagnosis of hydrocephalus.

The extracts and the compounds isolated from Phyllanthus amarus Schumm and Thonn (Family: Euphorbiaceae) have shown a wide spectrum of pharmacological activities including antiviral, antibacterial, antiplasmodial, antimalarial, antimicrobial, anticancer, antidiabetic, hypolipidemic, antioxidant, hepatoprotective, nephroprotective and diurectic properties. Background: This investigation was aimed at exploring the anxiolytic potential of Phyllanthus amarus standardized extracts and predict probable role of marker phyto constitutents. Objective and Methods: Three standardized extracts of Phyllanthus amarus plant viz. standardized aqueous extract of Phyllanthus amarus whole plant (PAAE), standardized methanolic extract of P. amarus leaf (PAME) and the standardized hydro-methanolic extract of P. amarus leaf (PAHME) were tested in the classical animal models of anxiety: Elevated plus-maze model and Light & Dark Exploration test. Results: The lower doses of the tannin rich extract (PAHME) of the P. amarus possess significant anxiolytic activity compared to lignin rich (PAME) and aqueous extracts (PAAE), while at a higher dose (400mg/kg) the results of all three extracts appears to be potentially sedative. While the molecular docking studies support these probable anxiolytic, the sedative effects of the Phyllanthus amarus extracts could be due to the interaction of tannins and lignans with the GABAbenzodiazepine receptor complex. Conclusion: The results of the present study indicate that the tannin-rich extract of the P. amarus may have potential clinical applications in the management of anxiety. It can be further studied for optimum dosage to be used as a future of anti-anxiety drug development or as a standardized Phytomedicine.

Background: Tumor heterogeneity imposes great challenges on cancer treatment. Cancer stem cells (CSCs) are a leading factor contributing to tumor occurrence. However, the mechanisms underlying the growth of thyroid cancer (TCHA) are still unclear. Methods: Key genes regulating the characteristics of THCA, such as stemness were identified by combining gene expressions of samples downloaded from the Cancer Genome Atlas (TCGA) and were used to establish an mRNA expression stemness index (mRNAsi) through machine learningbased methods. The relationships of mRNAsi, THCA clinical features and molecular subtypes were analyzed. Weighted Gene Co-Expression Network Analysis (WGCNA) was performed to obtain mRNAsi-related gene modules and determine mRNAsi-related differentially co-expressed genes. Key genes related to mRNAsi were screened by protein interaction network. Functional analysis was conducted and expressions of key genes were verified in multiple external data sets. Results: The mRNAsi score, which was found to be lower in the TCHA tissues than that in normal tissues (p<0.05), was positively correlated with a slow progression of tumor prognosis (p=0.0085). We screened a total of 83 differentially co-expressed genes related to mRNAsi and multiple tumor pathways such as apoptosis, tight junction, cytokine-cytokine receptor interaction, and cAMP signaling pathway (p<0.05). Finally, 28 protein interaction networks incorporating 32 genes were established, and 3 key genes were identified through network mining. 3 core genes were finally determined, as their low expressions were strongly correlated with the progression of THCA. Conclusion: The study found that NGF, FOS, and GRIA1 are closely related to the characteristics of THCA stem cells. These genes, especially FOS, are highly indicative of the prognosis of THCA patients. Thus, screening therapy could be used to inhibit the stemness of TCHA.

Aim: The aim of this study was to investigate the efficacy of thiol disulfide homeostasis and Ischemia Modified Albumin (IMA) values in predicting the technical difficulties that might be encountered during laparoscopic cholecystectomy. Materials and Methods: The study included 65 patients who underwent laparoscopic cholecystectomy due to cholelithiasis at the General Surgery Clinic of Ankara Numune Training and Research Hospital. All patients’ demographic data, previous history of cholecystitis, a history of chronic illness, preoperative white blood count (WBC), liver function tests (AST, ALT), amylase and lipase levels, intra-operative adhesion score, the ultrasonographic appearance of gall bladder, duration of hospital stay, duration of operation, thiol disulfide and IMA values were evaluated. Results: Native thiol and total thiol averages were higher in patients without a history of cholecystitis, and on the other hand, disulfide, disulfide/native thiol rate, disulfide/total thiol rate, native thiol/total thiol rate and IMA averages were higher in patients with a history of cholecystitis. While there was a statistically significant negative correlation between native and total thiol values and age, duration of surgery and duration of hospital stay; IMA, disulfide, disulfide/Total thiol, Native/Total thiol and disulfide/Native thiol rates were higher in older patients with a longer duration of surgery and hospital stay. In addition, preoperative IMA, disulfide, disulfide/Total thiol, Native/Total thiol and disulfide/Native thiol were observed to increase as the degree of intraoperative pericholecystic adhesion increased. Conclusion: We believe that the evaluation of thiol disulfide homeostasis and IMA parameters prior to laparoscopic cholecystectomy can be used as an effective method for predicting intraoperative difficulties.

Background: Coronavirus Disease 2019 (COVID-19) pandemic continues to threaten patients, societies and healthcare systems around the world. There is an urgent need to search for possible medications. Objective: This article intends to use virtual screening and molecular docking methods to find potential inhibitors from existing drugs that can respond to COVID-19. Methods: To take part in the current research investigation and to define a potential target drug that may protect the world from the pandemic of corona disease, a virtual screening study of 129 approved drugs was carried out which showed that their metabolic characteristics, dosages used, potential efficacy and side effects are clear as they have been approved for treating existing infections. Especially 12 drugs against chronic hepatitis B virus, 37 against chronic hepatitis C virus, 37 against human immunodeficiency virus, 14 anti-herpesvirus, 11 anti-influenza, and 18 other drugs currently on the market were considered for this study. These drugs were then evaluated using virtual screening and molecular docking studies on the active site of the (SARS-CoV-2) main protease (6lu7). Once the efficacy of the drug is determined, it can be approved for its in vitro and in vivo activity against the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which can be beneficial for the rapid clinical treatment of patients. These drugs were considered potentially effective against SARS-CoV-2 and those with high molecular docking scores were proposed as novel candidates for repurposing. The N3 inhibitor cocrystallized with protease (6lu7) and the anti-HIV protease inhibitor Lopinavir were used as standards for comparison. Results: The results suggest the effectiveness of Beclabuvir, Nilotinib, Tirilazad, Trametinib and Glecaprevir as potent drugs against SARS-CoV-2 since they tightly bind to its main protease. Conclusion: These promising drugs can inhibit the replication of the virus; hence, the repurposing of these compounds is suggested for the treatment of COVID-19. No toxicity measurements are required for these drugs since they were previously tested prior to their approval by the FDA. However, the assessment of these potential inhibitors as clinical drugs requires further in vivo tests of these drugs.

Background: One of the principal factors in the field of research in green chemistry is to drive chemical reactions using ultrasonication as a versatile synthetic tool. Moreover, nanostructured metal salts occupy an important position as low cost, efficient, heterogeneous, and green catalysts in chemical reactions. Pyrimidine has also acquired significance because it is a core structure in a variety of natural and non-natural agents, many of which display versatile biological activities and medical applications. Objective: The aim of this study was to explore the role of nickel(II) chromite nanoparticles (NiCr2O4 NPs) as a green and recyclable catalyst for the synthesis of 2,4-diamino-6-arylpyrimidine- 5-yl cyanides under ultrasonic radiation. Methods: A direct cyclocondensation reaction of guanidine nitrate, aromatic aldehydes, and malononitrile was performed using NiCr2O4 NPs as an effective heterogeneous catalyst under ultrasonic radiation at room temperature conditions to prepare 2,4-diamino-6-aryl-pyrimidine-5-yl cyanides in high yields. The described catalyst was prepared successfully according to a simple hydrothermal route and fully characterized by the X-Ray diffraction (XRD) technique, dispersive energy X-Ray (EDS) analysis, scanning electron microscopy (SEM), and dynamic light scattering (DLS). Results: A number of 2,4-diamino-6-aryl-pyrimidine-5-yl cyanides were effectively synthesized in high yields (94-98%) within short reaction times (15 min). All synthesized compounds were well characterized by IR, ¹H and ¹³C NMR spectroscopy, and also by elemental analyses. Conclusion: In conclusion, a simple, efficient, and green synthesis of 2,4-diamino-6-arylpyrimidine- 5-yl cyanides was developed using NiCr2O4 NPs as a green nanocatalyst, and under ultrasound radiation as a green tool. The mild reaction conditions, avoiding the use of toxic solvents or reagents, high atom economy, high yields, and simple workup are the attractive features of this new protocol.

Aims & Objective: Armoracia rusticana has high medicinal values and is an excellent source of phytochemicals. This study was aimed to evaluate the antidiabetic potential of bioactive compounds from Armoracia rusticana. Methods: The antidiabetic analysis revealed that Armoracia rusticana was highly active against α glucosidase with IC50 values of 5.6 μg/ml. Furthermore, molecular docking was used to identify the active constituents against α-glucosidase, while using acarbose as a controlled drug. Results: Upon phytochemical screening, it was found that six out of ten phytochemicals were successfully docked in the respective binding sites. The lead phytochemical was Quercetin 3-Obeta- D-xylopyranoside, which displayed a more binding score as compared to acarbose. They were subjected to analyze for drug-like properties, which further strengthen its validation. Conclusion: It was, therefore, concluded that Armoracia rusticana might potentially be used in the amelioration of type 2 diabetes. Potential molecules identified from this study could be considered as a lead drug to cure diabetes mellitus.

Background: Cervical cancer (CESC), which threatens the health of women, has a very high recurrence rate. Purposes: This study aimed to identify the signature long non-coding RNAs (lncRNAs) associated with the prognosis of CESC and predict the prognostic survival rate with the clinical risk factors. Methods: The CESC gene expression profiling data were downloaded from TCGA database and NCBI Gene Expression Omnibus. Afterwards, the differentially expressed RNAs (DERs) were screened using limma package of R software. R package “survival” was then used to screen the signature lncRNAs associated with independently recurrence prognosis, and a nomogram recurrence rate model based on these signature lncRNAs was constructed to predict the 3-year and 5-year survival probability of CESC. Finally, a competing endogenous RNAs (ceRNA) regulatory network was proposed to study the functions of these genes. Results: We obtained 305 DERs significantly associated with prognosis. Afterwards, a risk score (RS) prediction model was established using the screened 5 signature lncRNAs associated with independently recurrence prognosis (DLEU1, LINC01119, RBPMS-AS1, RAD21-AS1 and LINC00323). Subsequently, a nomogram recurrence rate model, proposed with Pathologic N and RS model status, was found to have a good prediction ability for CESC. In ceRNA regulatory network, LINC00323 and DLEU1 were hub nodes which targeted more miRNAs and mRNAs. After that, 15 GO terms and 3 KEGG pathways were associated with recurrence prognosis and showed that the targeted genes PTK2, NRP1, PRKAA1 and HMGCS1 might influence the prognosis of CESC. Conclusion: The signature lncRNAs can help improve our understanding of the development and recurrence of CESC and the nomogram recurrence rate model can be applied to predict the survival rate of CESC patients in clinical practice.